Phosphoinositide Domains and Gradients in Asymmetric Lipid Bilayers: Physical Properties and Protein Recognition

脂类是一类生物分子，可以影响甚至控制细胞膜上发生的几乎所有过程。它们对细胞生理学和人类生物学非常重要。生物膜是脂类的双层。双层的两个部分具有不同的总体脂组成以及不同的侧向组成。每个部分都包含具有不同脂组成和物理性质的结构域。不同的蛋白质被吸引到这些区域。因此，脂质和蛋白质形成超分子结构，是大量重要细胞功能的平台，最显著的是细胞信号。通过这一奖项，化学部的生命过程化学计划和分子和细胞生物科学部的分子生物物理学计划将资助伍斯特理工学院的Arne Gericke博士研究生物膜模拟中的结构域。更具体地说，Gericke研究小组调查了位于两个相对的双层中的域是否彼此对齐(注册)，如果是的话，位于注册中的两个域是否在物理上相互影响。本科生、研究生和博士后参与了这项研究。本科生有机会在德国多特蒙德工业大学参加为期10周的暑期研究体验。Gericke博士继续为中学生开发和教授夏令营，向学生介绍水作为所有生命的基础的独特性质，洗涤剂和脂类的性质，以及生物膜的独特和重要性质。磷脂酰肌醇(PIPs)脂类可影响多种蛋白质功能。PIP介导的信号转导的特异性植根于脂质头基的化学性质，它介导与不同蛋白质基序的特定相互作用。信号的时间控制是由磷酸化或去磷酸化PIP的激酶和磷酸酶提供的。PIP介导的信号转导的空间控制源于PIP在细胞膜上横向分布的不均匀，这导致了PIP富集域和梯度的产生。木筏是富含鞘脂和胆固醇的液体有序结构域，位于质膜的外叶中，影响内叶上的信号事件。许多PIP介导的信号事件与脂筏相关；然而，鉴于PIP的化学性质，它们有望分割成液膜补片而不是脂筏。目的1通过制备不对称脂质双层来解决这个长期存在的难题，该双层在外叶上显示脂筏混合物，而在内叶上显示含有PIP的脂类混合物。Gericke博士的团队使用这些不对称的脂类双层来确定导致域名注册的条件。目的2研究三种外周膜蛋白Marcks、PTEN和Akt如何与三种类型的PIP结构域(液体PIP/胆固醇、液体有序PIP/胆固醇以及与相对叶中的RAFT结构域接触的PIP/胆固醇)相互作用。Marcks与PIPs的相互作用是非特异性的，纯粹是静电的，PTEN与磷脂酰肌醇-4，5-二磷酸结合，Akt蛋白有一个口袋与磷脂酰肌醇-4，5-二磷酸结合。脂质梯度对于重要的细胞过程是至关重要的，包括胞质分裂和定向细胞运动。尽管基于细胞的研究强调了脂质梯度对细胞功能的重要性，但尚未解决脂质梯度的物理化学基础。Gericke博士的团队开发了一种微流控脂质梯度发生器，以系统地研究脂质梯度如何相互作用和/或脂质双层上方水相中的蛋白质梯度如何创建和维持脂质梯度。在目标3中，Gericke博士研究了PI3K和PTEN的相反梯度如何产生血脂梯度，以及预先存在的区域对梯度发展的影响。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Lipids are a class of biomolecules that affect or even control virtually all processes that occur on cellular membranes. They are very important for cell physiology and human biology. Biological membranes are bilayers of lipids. The two parts of the bilayer have different overall lipid composition as well as different lateral composition. Each part contains domains that have distinct lipid compositions and physical properties. Different proteins are attracted to these domains. As a result, lipids and proteins form supramolecular structures that are platforms for a large number of important cellular functions, most notably cell signaling. With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences are funding Dr. Arne Gericke from Worcester Polytechnic Institute to investigate the domains in biological membrane mimics. More specifically, the Gericke research group investigates whether the domains situated in the two opposing bilayers line up ("register") with each other and if so, whether the two domains situated in register physically affect each other. Undergraduate, graduate and postdoctoral students participate in the research. The undergraduate students have the opportunity to participate in a 10-week summer research experience at the Technical University Dortmund, Germany. Dr. Gericke continues to develop and teach a summer camp for middle school students that introduces the students to the unique properties of water as the foundation for all life, to the properties of detergents and lipids, and to the unique and important properties of biological membranes. Phosphoinositide (PIPs) lipids affect an extraordinary variety of protein functions. The specificity of PIP-mediated signaling is rooted in the chemical properties of the lipid's headgroup, which mediates specific interactions with distinct protein motifs. Temporal control of signaling is provided by kinases and phosphatases that phosphorylate or dephosphorylate PIPs. The spatial control of PIP-mediated signaling stems from the non-uniform lateral distribution of PIPs in the cell membrane, which gives rise to PIP-enriched domains and gradients. Rafts, which are liquid-ordered domains enriched in sphingolipid and cholesterol, are found in the outer leaflet of the plasma membrane and affect signaling events on the inner leaflet. Many PIP-mediated signaling events have been associated with lipid rafts; however, given the chemical nature of PIPs, they are expected to partition into fluid membrane patches rather than rafts. Aim 1 addresses this long-standing conundrum by fabricating asymmetric lipid bilayers that exhibit a lipid-raft mixture on the outer leaflet and PIP-containing lipid mixtures on the inner leaflet. Dr. Gericke's group uses these asymmetric lipid bilayers to determine the conditions that result in domain registration. Aim 2 explores how three peripheral membrane proteins, MARCKS, PTEN, and Akt, interact with three types of PIP domains (fluid PIP/cholesterol, liquid-ordered PIP/cholesterol, and PIP/cholesterol in contact with a raft domain in the opposing leaflet). The MARCKS interactions with PIPs are non-specific, purely electrostatic, PTEN binds phosphatidylinositol-4,5-bisphosphate, and the Akt protein has a pocket to which phosphatidylinositol-4,5-bisphosphate binds. Lipid gradients are crucial for important cellular processes, including cytokinesis and directed cell movement. While cell-based studies have highlighted the importance of lipid gradients for cellular function, the physiochemical underpinnings of lipid gradients have not been addressed. Dr. Gericke's group has developed a microfluidic lipid gradient generator to systematically study how lipid gradients interact and/or protein gradients in the aqueous phase above the lipid bilayer create and maintain lipid gradients. In aim 3, Dr. Gericke investigates how opposing gradients of PI3K and PTEN create lipid gradients and the effect of pre-existing domains on gradient development.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.